The present invention relates to the field of methods and devices for preparing samples for chemical analysis. This invention relates more particularly to equilibrium dialysis cells and a dialysis of a unique configuration and efficiency for assaying biological samples, as well as assaying biological samples with an automated pipettor.
There are many existing assays that measure the concentration of a desired molecule or ion in solution, referred to herein to as an analyte. For example a user might want to test for the concentration of the analytes thyroxine, estradiol or testosterone in a blood sample.
There are a wide variety of chemical analytic techniques used to detect analyte, such as chromatography, chemoluminesce, radioimmunoassay (RIA), Enzyme Immunoassay, Enzyme-Linked-ImmunoSorbent-Assay (ELISA) and flow techniques. These tests typically attach a marker to the analyte and then test for the presence of the marker to determine the presence of the analyte itself.
It is difficult however to measure analyte directly from a raw sample because in many instances the analyte is found in the sample as a solution of both free analyte and bound analyte. Bound analyte is usually bound to a high-molecular weight protein, and is a type of interference. An interference will react to the assay in the same manner as will the free analyte. More generally an interference is a substance, other than the material desired to be assayed, that also responds to the chosen analytical method thereby distorting the results, or a material that can prevent the assayed material from being measured at all.
In addition to the bound analyte there are other interferences commonly found in a raw sample. The sample may contain impurities such as larger molecules or solid components which can lead to adulterated analysis results. Impurities which commonly occur in sampling are, for example, extraneous proteins found when analyzing food; filter fibers and dust particles are commonly a problem when analyzing environmental samples.
When samples of assays contain interferences the results must be arrived at by analog means. For example, when there is bound analyte acting as an interference, analog means are arrived at by correlating the apparent result with standardized models to calculate an approximation of free analyte found in a given sample. The amount of free analyte present in the solution is estimated based on a characteristic ratio of bound analyte to free analyte found in past tests using the same technique for the same analyte.
Analog testing is essentially guesswork and at best is only an estimate of the free analyte. Analog determination is further complicated and unreliable because the ratio of free analyte to bound analyte may vary depending on the disease state of the patient, medications the patient is taking, etc.
Interferences can first be separated from the raw sample to produce a more accurate direct measurement. Equilibrium dialysis can be used for purifying a raw sample to extract the analyte to be assayed. The purified free analyte is separated from the bound analyte and other impurities and then measured alone. A dialysis cell and method of analyte separation is the subject of the present invention.
A problem with the use of equilibrium dialysis is the relatively long time it takes for the dialysis reaction to take place.
Dialysis is the separation of suspended colloidal particles in solution, the retentate, from the dissolved analyte ions or molecules of small dimensions. This separation is achieved by taking advantage of their unequal rates of diffusion through the pores of a semipermeable membrane. Equilibrium dialysis takes place across a semipermeable membrane by action of osmotic pressure. A semipermeable membrane is placed between a raw sample held in one chamber and an acceptor solution, the dialysate, held in a second chamber. The dialysate is a dialysis buffer solution that is chemically compatible with a given retentate and analyte. These two chambers together with the semipermeable membrane comprise a basic dialysis cell.
The permeability of the membrane is designed such that the analytes can migrate through the semipermeable membrane but the retentate and other interferences are excluded from migrating through to the dialysate. The retentate and other interferences are typically of a larger molecular weight and the semipermeable membrane allows only those molecules of lower molecular weight to migrate. The size or weight at which the largest molecule can migrate through a given semipermeable membrane is termed the Molecular Cut-Off Weight (MWCO).
Separation by dialysis is a slow process, the rate of dialysis depending in part on the differences in particle size and diffusion rates between the analyte and the non-analyte constituents. The rate at which the dialysis occurs depends on several other factors, some of which are the ratio of the analyte molecular weight to the membrane MWCO, the surface area of the membrane mutually contacted by the sample and the dialysate, the temperature of the two solutions and the amount of diffusion of substances that must first occur within both solutions for total equilibrium to be reached.
The diffusion that must take place in order for the dialysis to proceed to final equilibrium is slowed when the ratio of the membrane surface area that contacts either the retentate or the dialysate is small compared to the volume of its respective chamber. The reaction may also be impeded by molecules of the retentate and other interferences blocking the pores of the semipermeable membrane.
The analytes migrate through the membrane into the acceptor dialysate solution until an equal concentration on both sides of the membrane is established. As the dialysis process occurs, concentration gradients on either side of the membrane limit the rate of migration of analyte. The net migration of the analyte is directed toward the dialysate chamber and takes place as long as the concentration of the analytes in the sample chamber is larger than in the dialysate. At equilibrium the concentration of the analytes in the dialysate comprises a value identical to that of the retentate sample.
Current methods and devices used for dialysis require very long dialysis times to reach equilibrium, in some instances 17 hours or more.
One type of dialysis cell utilizes reusable blocks with injection ports in the sides to inject the sample or the dialysate. Membranes are placed between the blocks and dialysate and sample are alternately injected into the reusable blocks through the ports. A series of blocks may be sandwiched together in an alternating block/membrane/block fashion.
As used herein a first dialysis solution means either the sample or the dialysate. A second dialysis solution means the sample or dialysate as well, but the one of these two solutions not selected as the first dialysis solution.
The dialysis cell of the Nelson U.S. Pat. No. 4,963,256 utilizes an outer chamber containing a first dialysis solution. There is also provided an inner chamber which containing a second dialysis solution. The end of the inner cylindrical chamber is covered by a semipermeable membrane and inserted within the outer container. Because the semipermeable membrane of the Nelson cell covers only the end of a cylindrical member however, only about 90 mm2 of surface area of semipermeable membrane is available to mutually contact both of the dialysis solutions for dialysis.
In addition to the great amount of time it takes to allow both liquids to equilibrate, existing dialysis cells generally cannot be readily used with automated laboratory equipment such as an automatic pipettor. In most cases a dialysis cell must be individually loaded with the sample and the dialysate solution, requiring time and expensive manual labor to accomplish.
What is needed then is a dialysis cell that can take advantage of one or more of the variables in dialysis cell design to reduce the time that it takes for dialysis to reach equilibrium. What is also needed is a method to use a dialysis cell to shorten equilibrium time.
What is also needed is a dialysis cell that can be used with automated pipetting equipment to both increase the number of dialysis assays that can be done as well as to reduce the expense of manual labor to perform these tests.
It is therefore a first object of the present invention to provide a dialysis cell of a design that affords the maximum available semipermeable membrane surface area in mutual contact with both the analyte and the dialysate solution.
A second object of this invention is to minimize the time of reaching equilibrium in a dialysis cell by maximizing the surface area of the semipermeable membrane relative to the volume of the solutions used to perform the dialysis, thereby reducing the time needed for diffusion of the solutions during dialysis.
A third object of this invention is to provide a dialysis cell and method for its use that will allow using the dialysis cell with an automated pipettor.
The dialysis cell of the present invention encompasses a substantially rigid elongate outer container in the general shape of a test tube, the outer container having an open end and a closed end. There is also provided a substantially rigid elongate inner container, also having an open end and a closed end and sized to fit inside of the outer container and conform closely to the shape of the outer container.
The inner container centrally displaces liquid placed in the outer container when it is inserted therein, causing the liquid to envelope in the inner container. The space comprising the volume of the inner container is centrally displaced by a displacing member, which is comprised of either the design of the inner container itself, for example a portion of the closed end of the inner container being centrally inverted into the inner container, or, for example, is comprised of a second member placed in the inner container, such as a plunger to centrally displace the liquid of the inner container. This central displacement of the liquid in the inner container causes the liquid in the inner container to come into greater contact with the inner wall of the inner container and therefore also a semipermeable membrane contoured on or around the inner wall of the inner container.
The lengthwise portion of the elongate inner container wall incorporates a semipermeable membrane through which dialysis takes place. When the liquids of both the outer container and the inner container are centrally displaced they are brought into greater mutual contact with the semipermeable membrane.
In the preferred embodiment the incorporated semipermeable membrane is provided by one or more fluid permeable apertures in a portion of the lengthwise wall of the inner container. The apertures are covered with semipermeable membrane. the fluid permeable apertures in a portion of the lengthwise wall of the inner container are first covered with porous mesh for structural support of the semipermeable membrane, the semipermeable membrane is applied over the porous mesh.
The invention takes advantage of the additional membrane surface area that may be utilized by placing semipermeable membrane along the length of the inner container. The closed end of the inner container may be additionally provided with apertures and semipermeable membrane for creating additional available surface area. This design provides more available membrane surface area while maintaining the advantages of having a substantially rigid tube that may be used in an automated pipettor machine. The automated pipettor machine may be used to move the dialysis cell, to introduce a dialysis solution to the inner container or the outer container, or to remove a dialysis solution from the inner container or outer container.
A first dialysis solution is placed in the outer container, the serum retentate sample is used in the preferred embodiment, then the inner container is inserted into the outer container. A second dialysis solution is placed within the inner container, the dialysate in the preferred embodiment, creating a dialysis cell. The two containers are attached to each other to hold them in fixed relation.
The open end of the inner container and the open end of the outer container are designed to be threadably attached after the inner container is inserted into the outer container, holding the two containers in fixed relation to each other. Alternatively for example, the inner container may be inserted into the outer container and a threaded cap affixed to the outer container, holding the inner container in. The inner container and the outer container may be attached in any convenient way, for another example by attaching the open ends of the two containers with a snap-lock to each other, with complementary snap-lock flanges on the open end of each container. These designs are well known in the art and are presented by way of example and are not exhaustive of the alternative ways to attach the open ends of the two containers.
In a first embodiment of the dialysis cell the closed end of the inner container is concave and centrally inverted into the inner container in order to centrally displace the solution within the inner container and cause the available solution to come into greater contact with the surface area of the wall of the inner container incorporating the semipermeable membrane. Centrally displacing the solution in this manner reduces the volume of solution needed to inundate the available semipermeable membrane and the decrease in volume also decreases the time needed for any intra-solution diffusion to take place.
A portion of the wall running along the length of the inner container, excluding the inverted portion, has apertures to allow fluid communication between the inner container and the outer container. The apertures of the inner wall of inner container are first covered with porous mesh to provide support for semipermeable membrane, then a semipermeable membrane is placed over the apertures on the outer wall of the inner container. Use of the porous mesh is of course not necessary if a given semipermeable membrane does not require the additional structural support of the porous mesh because the apertures are small enough or the membrane is strong enough in such properties as tensile and shear strength to cover the apertures by itself without tearing. The porous mesh may also be placed on the outside wall of the inner container over the apertures, then the semipermeable membrane placed thereover.
The outer container is supplied with a first dialysis solution, then the inner container is supplied with a second dialysis solution and placed in the outer container. The outer container and the inner container are attached at their respective open ends, holding the two containers tightly with respect to one another. In this embodiment the inner container may also have a thin puncturable film covering the open ends of both containers.
The dialysis reaction is then allowed to proceed to equilibrium. A quantity of the dialysate is taken after equilibrium and subjected to one of the above-recited analyses to determine serum concentration of the analyte.
In a second embodiment an elongate outer container is provided, having an open end and a closed end. There is also provided an inner container that closely conforms to the outer container, the inner container having an open end and a closed end. A wall portion of the inner container between the open end and the closed end has apertures to allow fluid communication between the inner container and the outer container.
In the preferred embodiment the inner wall of the inner container having apertures is covered with porous mesh to provide support for a semipermeable membrane, then a semipermeable membrane is secured over the apertures on the outer wall of the inner container. The outer container is then supplied with a first dialysis solution. The inner container is supplied with a second dialysis solution and placed in the outer container, creating a dialysis cell. The outer container and the inner container are attached holding the two tubes in fixed relation to each other.
A plunger is then inserted into the inner container to drive the dialysis solution of the inner container up the length of the interior of the inner container. The porous mesh acts to guide the plunger to be centrally located within the inner container, maintaining a uniform distance of about 250 xcexcm between the plunger and the interior wall of the inner container. This configuration results in a shorter equilibrium dialysis time because there is both a greater area of semipermeable membrane in mutual contact with both dialysis solutions and the time necessary for diffusion is greatly reduced because there is a smaller volume of ambient dialysis solution within which any diffusion of an analyte has to take place.
In the above manner dialysis time can be substantially reduced from the current average of, for example, seventeen hours. The new design also allows freedom from human intervention because the dialysis cells can be prepared in advance with dialysate buffer solution placed within the outer container. The user simply adds the sample to the inner container and allows the dialysis to go to equilibrium. After reaching equilibrium the user removes the inner container and inserts a probe into the prepared tube of dialysate.
Alternatively, the inner container may be prepared in advance by being filled with dialysate. The user then adds the sample to the outer container and inserts the inner container.
These new designs can also be used with automated equipment because they are in the general shape of a test tube and require only the insertion of a quantity of sample into one of the containers.
Additional aspects and advantages of the present invention are set forth in the following description and claims, particularly when considered in conjunction with the accompanying drawings in which like parts bear like reference numerals.